Histology: Finished up the histology slide images of Oly and gigas gonad tissue. I uploaded the images into folders in the GitHub repository here. Additionally, I sent off the next batch of histology cassettes to be made into histology slides.
Oysters: Fed the oysters in the tank algae paste. Let sit for 1 hour. Measured salinity: 30. Started a feed log in order to keep track of salinity and feeding schedule.
Hooray! PBJelly actually finished. Not sure what happened to the downtime that was supposed to be occurring yesterday and today, but apparently it didn’t interfere with things.
I’ve copied what I suspect to be the output files from Hyak to here.
I’m going to try and copy all of the output files from Hyak over to Owl, but first I have to clean up the ~2.75mm assembly temp files that PBJelly decided to not clean up…
Skyline works with our demultiplexed files!
Emma sent over a test .blib made from my demultiplexed and converted oyster 1 mzML file. Using the new Windows machine, I ran this file through Skyline Daily. I followed settings Emma suggested as well as the protocol from my previous Skyline test.
Peptide digestion settings
Adding my background proteome.
My background proteome FASTA file can be found here.
Modifying my peptide digestion settings.
For digestion enzyme, I used Trypsin.
Modification and Quantification.
Last time I used Skyline, I did not touch these sections.
Adding FASTA file
I imported the FASTA file like I did previously, but it did not work.
Error message from my attempt to import my FASTA file:
I decided to move on and change my transition settings before I figured out how to add my protein sequences.
Adding protein sequences
I opened up my FASTA file in Notepad and literally copied and pasted my sequences into the lefthand bar. It worked!
Importing my raw files as “Results”
Added oyster 1 to ensure it worked. It did!
I then imported the rest of my files.
Now that my Skyline file is set up, all I need to do is replace the .blib with one from all of my mzML files and start picking targets for SRM.
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I re-ran the data files through a different trimming program, Trimmomatic, with a specified Illumina TruSeq adapter set and got better results from FastQC. Running things through Bismark once more, and we’ll see if we get better mapping rates that way. Tomorrow I’ll likely run stuff through BSMap as well to see if I can match, or beat the ~20% rate Steven got comparing to the gigas genome.
New MultiQC report
I ran Bismark on the oil exposed C. virginica samples from LSU over the weekend and got pretty abysmal mapping rates, between 4% and 8% terrible. I looked through the quality data and noted that there was some oddities in one of the samples with residual TruSeq adapters post trimming.
I talked to Sam and he remembered that this was an issue with this data the last time it was looked at, so we decided to re-try the trimming function, specifying TruSeq adapters, as well as trimming the first 5 sequences due to relatively lower quality.
Notebook Entry 1 Copying, MD5 checking, and concatenating
Notebook Entry 2 First round of trimming
Notebook Entry 3 Bismark
Multi-QC report on initial trimming attempt.
I prepped the histology samples taken on 4/8 and 4/13 to be sent off for slide preparation. My samples are Ostrea lurida whole visceral mass, with the goal of analyzing gonad maturation, and Yaamini’s samples are exclusively Crassostrea gigas gonad. I sampled NF, SN & HL populations on 4/8 and K populations on 4/13. Megan, Grace, Kaitlin, and Rhonda helped me by sampling the NF, SN & HL groups, and I did the K groups myself.
Sequence of events and sample handling
- On 4/8 & 4/13: while sampling, once a cassette was full, it was immediately placed in the FIX solution.
- 24 hrs later the FIX was poured out of jars, and refilled with STABILIZER solution.
- On 4/20, to make sure we can read the correct orientation of the slides, I dotted the top left tissue in each cassette with blue ink. Here’s my setup, and images of all the samples within each cassette for reference.
#### I’ve also prepared a histology sample key.
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Just a quick update on some plumbing that needs to be done prior to getting my animals in buckets to spawn:
Several parts that I need were out of stock @ Harrington so I ordered them to be delivered to the hatchery- they’ll get there on Tuesday. This sets me back a few days. I’ll go out on Tuesday to do as much as I can, and then will hopefully finish up the gluing on Wednesday after class. At the earliest I’ll have animals in their spawning buckets on Thursday.
I’ve been watching the pH & temp via the http://ift.tt/2odpAq7 updates, and pH is hovering around 7.58, temp between 13.8-14.5. PSRF is lowering their heated line to 14degC on May 1st – since that’s 10 days away (and since my animals are currently at ~14) I think it’s wise to just condition them at 14.
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